Modified adenoviruses have proven convenient vector systems for investigative and therapeutic gene transfer applications, and adenoviral viral systems present several advantages for such uses. Such vectors can be produced in high titers (e.g., about 10.sup.13 pfu), and such vectors can transfer genetic material to nonreplicating, as well as replicating, cells (in contrast with, for example, retroviral vectors). The adenoviral genome can be manipulated to carry a large amount of exogenous DNA (up to about 8 kb), and the adenoviral capsid can potentiate the transfer of even longer sequences (Curiel et al., Hum. Gene Ther., 3, 147-154 (1992)). Additionally, adenoviruses generally do not integrate into the host cell chromosome, thus minimizing the likelihood that a recombinant adenovirus will interfere with normal cell function. Thus, aside from being a superior vehicle for transferring genetic material to a wide variety of cell types, adenoviruses represent a safe choice for gene transfer, a particular concern for therapeutic applications.
The development of adenoviral vectors rests on an understanding of viral genetics and molecular biology. Adenoviruses have diverging immunological properties and are divided into serotypes. Two human serotypes of adenovirus (Ad2 and Ad5) have been studied intensively, and their genomes have been completely sequenced. Other serotypes have been heavily studied as well, and it is apparent that all adenoviruses are structurally similar and that the overall organization of the adenoviral genome is conserved between serotypes such that specific functions are similarly positioned.
Structurally, all adenoviral virions are nonenveloped capsids with a number of surface proteins. Through interactions of these capsid proteins with the surface of a host cell, the virus is internalized and encased in an clathrin-coated organelle resembling an endocytotic vessel (Pastan et al., Concepts in Viral Pathogenesis, Notkins and Oldstone, eds. Springer-Verlag, New York. pp. 141-46 (1987)). The acidic condition within the vesicle alters the surface configuration of the virus, resulting in vesicle rupture and release of the virus into the cytoplasm of the cell, where it is partially freed of associated proteins while being transported to the nucleus.
Once an adenoviral genome is within the host cell nucleus, its expression proceeds through a highly ordered and well characterized cascade. Groups of adenoviral genes (i.e., translation units) are typically organized into common transcription units ("regions"), each having at least one distinct promoter. The transcript from each region is processed after transcription to generate the multiple MRNA species corresponding to each viral gene. Generally, the separate regions are either "early" or "late," although some genes are expressed as both early and late genes.
Cellular transcription factors first bind to the upstream enhancer of the first early (E1A) region of the adenoviral genome. The E1A gene products, in turn, regulate the expression of other early promoters, one of which (E2B) drives the expression of the transcription unit including three early genes involved in adenoviral DNA replication (Doefler, pages 1-95, in Adenovirus DNA, the Viral Genome, and Expression, Nojhoff, Boston, (1986)). These three proteins (the precursor terminal protein (pTP), the single-stranded DNA binding protein (ssDBP), and the DNA polymerase (poo) form a tight unit with at least three cellular proteins to drive priming and elongation of the viral genome (Bodnar et al., J. Virol., 63, 4344-53 (1989); Schnack et al., Genes Devel., 4, 1197-1208 (1990); Pronk et al., Clomosoma, 102, S39-S45 (1992); Kelly et al., pages 271-308, in The Adenoviruses (H. S. Ginsberg, ed.), Plenum Press, New York (1984)).
Once viral DNA replication commences, the activity of the early promoters declines (Sharp et al., pages 173-204, in The Adenoviruses (H. S. Ginsberg, ed.), Plenum Press, New York (1984)), as does the expression of cellular genes, due to the activity of the viral host shut-off early gene products. Conversely, promoters controlling the expression of the late genes become active beginning with the onset of viral DNA replication (Thomas et al., Cell, 22, 523-33 (1980)). Indeed, DNA replication appears necessary for the expression of some late genes. For example, while the major late promoter (MLP) exhibits some activity at early times, only the promoter proximal genes are expressed (Shaw et al., Cell, 22, 905-16 (1980); Winter et al., J. Virol., 65, 5250-59 (1991)). However, the activity of the MLP sharply increases following the onset of viral DNA replication (Shaw et al., supra), resulting in the expression of all the MLP gene products (Doeller et al., supra; Thomas et al., supra; Nevins et al., Nature, 290, 113-18 (1981)). Post-transcriptional processing of the major late transcription unit (MLTU) gives rise to five families of late mRNA, designated respectively as L1 to L5 which encode structural components of the viral capsid (Shaw et al., Cell, 22, 905-916 (1980)).
Adenoviral gene products are highly toxic to cells, and the components of the adenoviral capsid potentiate immune responses against infected cells (see, e.g., Yang et al., Proc. Nat. Acad. Sci. (USA), 91, 4407-11 (1994)). This immune response leads to tissue swelling and destruction of the transduced cells, shortening the period of time transgenes are expressed in the cells. Thus, "first generation" adenoviral vectors have been engineered to silence the adenoviral genome with the aim of reducing these deleterious effects. Because, as mentioned, the E1A gene products begin the cascade of viral gene expression, the earliest adenoviral vectors lacked hfunctional E1A regions. For example, insertion of an exogenous gene into the E1 region results in recombinant vectors that can express the exogenous gene but not the E1A gene. However, the recombinant adenoviruses must be propagated either in complementary cells or in the presence of a helper virus to supply the impaired or absent essential E1 products (Davidson et al., J. Virol., 61, 1226-39 (1987); Mansour et al., Mol. Cell Biol., 6, 2684-94 (1986)).
While such first generation (i.e., E1 deficient) viruses have proven effective in several gene transfer applications, they are not optimal for all uses. In particular, because they must be grown in the presence of E1-complementing DNA, at some frequency recombination events can generate a replication competent adenovirus (RCA). RCA contamination of viral stocks is problematic because RCAs can outgrow recombinant stocks and transform host cells. Moreover, at higher multiplicity of infections (m.o.i.s), several adenoviral promoters are active even in the absence of the E1A gene products, which can lead to the production of cytotoxic adenoviral proteins (Nevins, Cell, 26, 213-20 (1981); Nevins et al., Curr. Top. Microbiol. Immunol., 113, 15-19 (1984)). In turn, residual late gene expression can potentiate host immune responses eliminating virally transduced cells (see, e.g., Yang et al., supra; Gilgenkrantz et al., Hum. Gene. Ther., 6, 1265-74 (1995); Yang et al., J. Virol., 69, 2008-15 (1995); Yang et al., J. Virol., 70, 7209-12 (1996)).
Other than RCA generation, the disadvantages of first generation vectors are largely attributable to background expression of late gene products. One approach for blocking late gene expression is to selectively block viral replication by mutating the virus such that it fails to express one or more of the three E2B enzymes involved in viral DNA replication. However, while E1A-deficient viruses lacking E2B function can be generated, the approach requires the use of complementing cell lines or helper viruses to supply the missing essential gene product (Almafitano, J. Virol., 72(2), 926-33 (1998)). As discussed above, a major drawback to such an approach is that recombination events within packaging cells between such vectors and complementing genes can generate RCAs. Moreover, of the three E2B genes, it is not currently possible to propagate a virus lacking the ssDBP gene entirely because the required co-expression of the complementing ssDBP and E1A gene products in the same packaging cell is lethal (Klessig et al., Mol. Cell Biol., 4, 1354-62 (1984)). A vector has been constructed that has a temperature sensitive mutation in the ssDBP gene (Engelhardt et al., Proc. Nat. Acad. Sci. (USA), 91, 6196-6200 (1994)). In some systems, this vector has resulted in longer gene expression and reduced immune-inflammatory response than first generation vectors (Yang et al., Proc. Nat. Acad. Sci. (USA), 92, 7257-61 (1995); Engelhardt et al., Hum. Gene Ther., 5, 1217-29 (1994)). However, the temperature-sensitive mutation is imperfect, permitting some basal ssDBP activity at core body temperature, especially at high m.o.i.s (Yang et al., Proc. Nat. Acad. Sci. (USA), 92, 7257-61 (1995)). In addition to these drawbacks, the approach of disrupting the E2B region also impacts the MLTU because the three E2B genes lie on the chromosomal strand directly opposite the L1-L5 genes and the MLP, (see, e.g., Almafitano et al., Gene Ther., 4, 258-63 (1997)).
In view of the foregoing problems, there exists a need for a recombinant adenovirus, specifically a virus exhibiting reduced propensity to generate RCAs within packaging cells and less able than first generation vectors to express late viral gene products in a host cell.